Curbside particulate matter and susceptibility to SARS–CoV-2 infection

Background Biologic plausibility for the association between exposure to particulate matter (PM) less than 10 μm in aerodynamic diameter (PM10) and coronavirus disease 2019 (COVID-19) morbidity in epidemiologic studies has not been determined. The upregulation of angiotensin-converting enzyme 2 (ACE2), the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) entry receptor on host cells, by PM10 is a putative mechanism. Objective We sought to assess the effect of PM10 on SARS-CoV-2 infection of cells in vitro. Methods PM10 from the curbside of London's Marylebone Road and from exhaust emissions was collected by cyclone. A549 cells, human primary nasal epithelial cells (HPNEpCs), SARS-CoV-2–susceptible Vero-E6 and Calu3 cells were cultured with PM10. ACE2 expression (as determined by median fluorescent intensity) was assessed by flow cytometry, and ACE2 mRNA transcript level was assessed by PCR. The role of oxidative stress was determined by N-acetyl cysteine. The cytopathic effect of SARS-CoV-2 (percentage of infection enhancement) and expression of SARS-CoV-2 genes' open reading frame (ORF) 1ab, S protein, and N protein (focus-forming units/mL) were assessed in Vero-E6 cells. Data were analyzed by either the Mann-Whitney U test or Kruskal-Wallis test with the Dunn multiple comparisons test. Results Curbside PM10 at concentrations of 10 μg/mL or more increased ACE2 expression in A549 cells (P = .0021). Both diesel PM10 and curbside PM10 in a concentration of 10 μg/mL increased ACE2 expression in HPNEpCs (P = .0022 and P = .0072, respectively). ACE2 expression simulated by curbside PM10 was attenuated by N-acetyl cysteine in HPNEpCs (P = .0464). Curbside PM10 increased ACE2 expression in Calu3 cells (P = .0256). In Vero-E6 cells, curbside PM10 increased ACE2 expression (P = .0079), ACE2 transcript level (P = .0079), SARS-CoV-2 cytopathic effect (P = .0002), and expression of the SARS-CoV-2 genes' ORF1ab, S protein, and N protein (P = .0079). Conclusions Curbside PM10 increases susceptibility to SARS-COV-2 infection in vitro.

cigarette smoke extract (CSE) was also assessed because it is a source of oxidative stress, 15 it increases ACE2 expression in primary airway epithelial cells in vitro, 16 and ACE2 expression is increased in the lower airway of smokers. 17

Collection of PM 10
Curbside PM 10 was collected using a high-volume cyclone placed at a single site on the curbside of Marylebone Road, one of the most polluted roads in London. 18Sampling was done for 6 to 8 hours per day on 11 occasions.Diesel exhaust PM 10 was collected by placing the cyclone 0.1 m from the end of the tailpipe of an idling diesel car (BMW, model d330) for 15 minutes.The PM 10 was stored at room temperature in a sterile glass container.Aliquots of the diesel PM 10 and curbside PM 10 were diluted in Dulbecco PBS to a final concentration of 1 mg/mL and stored as a master stock at -208C.

CSE
CSE was prepared as previously reported. 19Briefly, cigarette smoke in filter material was prepared by using 3 cigarettes (0.8 mg of nicotine per cigarette; Marlboro, Philip Morris USA, Pittsburgh, Pa) "smoked" by a water aspirator, with the cigarette smoke aspirated through a cotton wool filter.CSE (100%) was obtained by vortexing the cotton wool filter in 2 mL of dimethyl sulfoxide diluted in medium.

Cells
The human AT2 epithelial cell line A549 was purchased from Sigma-Aldrich (Poole, UK) and maintained in Dulbecco modified eagle medium (DMEM) supplemented with FBS and penicillinstreptomycin (Lonza, Basel, Switzerland).The passage number was less than 20, with the same number of passages used in each set of experiments.Human primary nasal epithelial cells (HPNEpCs) were either obtained from a nonsmoking, nonvaping, healthy adult donor by using a dental brush (donor HPNEpCs) or purchased from PromoCell (Heidelberg, Germany).The nasal cells were maintained in airway epithelial cell growth medium (AECGM) by using a PromoCell supplement kit (Heidelberg, Germany) with Primocin (InvivoGen, San Diego, Calif) and stored cryogenically in freezing medium (AECGM: 10% FBS, 10% dimethyl sulfoxide) at passage 1.A vial of HPNEpCs was thawed and aliquoted into multiple T25 cell culture flasks (VWR International, Lutterworth, UK).The cells were maintained in AECGM until confluent.The passage number was less than 2. Vero-E6 cells were obtained from the American Type Culture Collection (ATCC, Middlesex, UK) and maintained in DMEM supplemented with FBS and penicillin-streptomycin (Lonza, Basel, Switzerland).The passage number was less than 20.Calu3 cells were purchased from ATCC maintained in Eagle minimum essential medium (ATCC) supplemented with FBS (ATCC) and penicillin-streptomycin (Lonza).The passage number was less than 5. Cell membrane integrity was assessed by lactate dehydrogenase (LDH) release, according to the manufacturer's instructions (Sigma-Aldrich).Treatment of cells with distilled water was used as a positive control and indicated 100% LDH release.For exposure to PM 10 , master stock PM 10 was thawed, thoroughly vortexed, and suspended at a final concentration of 1 to 20 mg/mL in Dulbecco PBS (2% FBS) containing a 2 3 10 5cell monolayer per well for 2 hours at 37 8C.Control samples of medium (ie, medium without without PM 10 ) were incubated with the same volume of Dulbecco PBS (2% FBS).In some experiments, the antioxidant N-acetyl cysteine was added with PM 10 at a final concentration of 5 mmol$L.

ACE2 expression by flow cytometry
Cells were washed twice and stained with either anti-ACE2 (Abcam, Cambridge, UK; catalog no.ab272690) or isotype control primary antibodies (Abcam; catalog no.Ab171870) for 1 hour at room temperature.The epithelial cell marker E-cadherin (Abcam; catalog no.Ab1416) was included in all reactions.The cells were then washed and stained with secondary antibodies conjugated to Alex Fluor 488 for ACE2/isotype expression (Abcam; catalog no.Ab150077), or allophycocyanin for Ecadherin expression (Abcam; catalog no.Ab130786) for 30 minutes in the dark at room temperature.The cells were finally washed, and ACE2 expression measured by using az BD FACS Canto II flow cytometer (BD Biosciences, San Diego, Calif).Ecadherin-positive cells were selected to exclude cell debris, and the mean fluorescence intensity of fluorescein isothiocyanate was calculated with adjustment for the isotypic control.

ACE2 expression by qRT-PCR
The cells were washed and total RNA was extracted by using the RNeasy mini kit (Qiagen, Manchester, UK) according to the manufacturer's instructions.cDNA was synthesized from total RNA by using Superscript II (Thermo Fischer cell culture flasks [VWR International]).Quantitative RT-PCR (qRT-PCR) was performed by using custom TaqMan array 96-well plates and TaqMan Fast Universal Master mix II with UNG (Life Technologies) with ACE2 and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Amplification was performed with the fast PCR 7500 (Life Technologies).The cycling conditions started with an initial activation step for 15 minutes at 958C, followed by 40 cycles of denaturation for 15 seconds at 948C, annealing for 30 seconds at 558C, and extension for 30 seconds at 708C, with fluorescence data collection performed during the extension step.The threshold cycle values were normalized to GAPDH, which is a frequently used reference gene, and relative quantification was analyzed by using the 2 -DDCt method.

Preparation and titration of authentic SARS-CoV-2
Wuhan Hu-1 strain SARS-CoV-2 (2019-nCoV/BavPat1/2020) authentic virus cell culture supernatant (isolate collection date January 1, 2020) was obtained from the European Virus Archive Global (EVAg, Charit e Universit€ atsmedizin Berlin, Germany).Virus stocks were prepared as previously described. 20,21Vero-E6 cells were seeded in 75-cm 2 cell culture flasks 24 hours before inoculation with virus cell culture supernatant containing 2.2 3 10 6 plaque forming units (PFU) in a volume of 10 mL of DMEM 10% FBS.The flasks were observed daily, and virus-containing cell culture medium was harvested when more than 80% of the cells showed CPE.The supernatant was centrifuged at 500 g for 5 minutes to clear cell debris and aliquots stored at -808C.To determine the titer of SARS-CoV-2 virus stocks, Vero-E6 cells were seeded in 48-well plates at a rate of 3310 4 cells per well.After 24 hours, adherent cell monolayers were challenged with serial 1:10 duplicate dilutions of virus, and titer was assessed after 20 hours by in situ intracellular staining to identify foci of infection.The cells were washed in PBS, fixed in ice-cold methanol-acetone (50:50), and virus antigen was stained for 1 hour at 378C by using sera from convalescent individuals diluted 1:2000 in PBS (1% FCS).The cells were washed a further 3 times in PBS and incubated with goat anti-human IgG Southern Biotech, Birmingham, Ala) diluted 1:400 in PBS (1% FCS) for 1 hour at 378C.After 3 further PBS washes, 300 mL of 0.5-mg/mL 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside chromogenic substrate (X-gal) in PBS containing 3 mM potassium ferricyanide, 3 mM potassium ferrocyanide, and 1 mM magnesium chloride was added to each well.The infected cells incubated at 378C stained blue within 1 to 4 hours after addition of substrate, and the clusters of blue cells were counted as foci of infection to determine the virus titer defined as focusforming units (FFU)/mL.All authentic SARS-CoV-2 propagation and infectivity assays were performed in a containment level 3 facility.

SARS-CoV-2 infection
Vero-E6 cells were suspended in DMEM-supplemented FBS and penicillin-streptomycin overnight in adherent 46-well cell culture plates at 378C.The cells were then washed and cultured with PM 10 or control medium for 2 hours before being thoroughly washed and then challenged with 3 3 10 4 FFU/mL of SARS-CoV-2 (the median tissue culture infectious dose) in duplicate and incubated at 378C for 48 hours to 72 hours.The surviving cells were fixed in 3.7% (vol/vol) formaldehyde and stained with 0.1% (wt/vol) crystal violet solution.The crystal violet stain was resolubilized in 1% (wt/vol) SDS.Absorbance readings were taken at 57 nm by using a CLARIOStar Plate Reader (BMG Labtech, Aylesbury, UK).The absorbance readings were normalized to SARS-CoV-2 virus-only control wells, and enhancement/inhibition in infection was presented as the percentage change in CPE (percent enhancement of infection).

Viral gene expression by qRT-PCR
qRT-PCR was performed by using the TaqMan 2019-nCoV Assay Kit v1 and TaqPath 1-Step RT-qPCR Master Mix (A47532 and A15299, Thermo Fisher).The kit includes 3 primer sets that target SARS-CoV2 genes' open reading frames (ORF) 1ab, spike (S) protein, and nucleocapsid (N) protein.A standard curve was generated with an input of 1.5 3 10 6 FFU/mL, and serial 10-fold dilutions of SARS-CoV-2 viral stock were made.Viral RNA was then extracted from the stock to generate a standard curve.The cells were challenged with SARS-CoV-2 (Wuhan Hu-1) for 48 hours before the cell supernatants were removed and viral RNA was extracted from the cell lysates.Amplification was performed with the fast PCR 7500 (Life Technologies).The cycling conditions started with an initial hold stage for 2 minutes at 258C, reverse transcription for 15 minutes at 508C, and an activation stage for 2 minutes at 958C, followed by 40 cycles of denaturation for 3 seconds at 948C and annealing/extension for 30 seconds at 608C, with fluorescence data collection being performed during the extension step.The threshold cycle values were normalized to the standard curve for quantification, and the results were expressed as FFU/mL.

Ethics
Sampling of nasal epithelial cells was approved by the Queen Mary University of London ethics committee (reference no.15/ NE/0237).

Statistical analysis
Data are summarized as medians and analyzed by either the Mann-Whitney U test or Kruskal-Wallis test with the Dunn multiple comparisons test.The data are from at least 4 separate experiments.The analysis were performed by using Prism 9.0, with P values less than .05considered statistically significant.Statistical analysis was performed by using GraphPad Prism 9.0 (GraphPad Software, La Jolla, Calif).

DISCUSSION
In this study, expression of ACE2 (the receptor coopted by SARS-CoV-2 to infect cells 22 ) by human nasal epithelial cells was stimulated by PM 10 collected from the curbside of a London main road dominated by diesel traffic. 23PM 10 stimulation of ACE2 expression was also found in A549 cells, a cell line that is widely used to model AT2 cell responses 24 and normally expresses very low to absent ACE2. 10 Consistent with these A549 data, Sagawa et al 11 reported increased ACE2 protein expression in murine AT2 cells after intratracheal instillation of 500 mg of urban PM, 11 Kim et al 13 reported increased ACE2 expression by human pluripotent stem cell-derived airway cells cultured in vitro with 50 mg/mL diesel PM 2.5 for 48 hours, and Zhu et al 12 reported increased ACE2 protein expression in human pulmonary alveolar epithelial cells exposed to 50 mg/mL of urban PM for 24 hours.In the present study, the functional relevance of PM 10 -stimulated ACE2 was established in Vero-E6 cells.In this SARS-CoV-2 trophic cell line, curbside PM 10 increases ACE2 expression and ACE2 transcript levels and enhances SARS-CoV-2 infection and SARS-COV-2 viral gene expression.
To date, the pathway by which PM 10 simulates ACE2 expression is unclear.However, Santurt un et al 25 speculated that the initial stimulus is PM-induced oxidative stress.Compatible with this speculation, we found that the antioxidant N-acetyl cysteine attenuated PM 10 -stimualted ACE2 expression.Furthermore, we found that CSE, a known source of oxidative stress, 15,16 increased ACE2 expression in both A549 cells and HPNEpCs.Indeed, in HPNEpCs, CSE 5% was the more potent stimulus of ACE2 expression.We therefore speculate that smokers living in areas with high concentrations of PM 10 are more vulnerable to SARS-CoV-2 infection.
There are limitations to this study.First, because the PM concentration in nasal epithelial lining fluid is unknown, whether exposure of cells to 10 mg/mL PM 10 in vitro reflects exposure in vivo is unclear even though concentrations of PM 10 on the curbside of Marylebone Road may exceed 180 mg/m 3 . 26In addition, Zielinski et al 27 have calculated that 50 mg/mL of PM in in vitro experiments likely reflects PM concentrations in the human airway epithelial lining fluid when individuals are exposed to the maximum UK hourly average value.Second, the SARS-CoV-2 infection experiments were performed in nonhuman Vero-E6 cells because in contrast to primary airway epithelial cells, this cell line is permissive for SARS-CoV-2 infection, resulting in a highly reproducible CPE.Third, we did not assess the expression of other proteins that support SARS-CoV-2 infection.For example, SARS-CoV-2 uses transmembrane protease serine 2 (TMPRSS2) to prime its spike protein, although Vero-E6 cells express TMPRSS2 at very low levels. 8Fourth, the demographic details of the purchased human primary nasal epithelial cell donor are unknown.However, we replicated the ACE2 response of purchased cells by using primary nasal cells from a local adult donor.Fifth, Crilley et al 28 found elements in PM from Marylebone Road that are indicative of abrasion processes from tyres and brakes.Although we cannot exclude upregulation of ACE2 by PM from these noncombustion sources, diesel PM is likely to be a major source of curbside PM 10 in the present study because when using particle-induced x-ray emission analysis, Crilley et al 28 found that diesel emissions are the main source of curbside PM on Marylebone Road, and we confirmed that diesel PM 10 per se upregulates ACE2 expression.Finally, whether PM 10 increases susceptibility of nasal epithelial cells to other infections is unclear.However, Takizawa et al 29 reported that diesel exhaust PM upregulates intercellular adhesion molecule-1 (ICAM-1), the entry receptor for rhinoviruses, 30 in human bronchial epithelial cells in vitro.
In summary, PM 10 sampled from the curbside of a main road in London via induction of oxidative stress upregulates ACE2 expression by airway epithelial cells in vitro.This in turn increases vulnerability to SARS-CoV-2 infection.Thus, the association between exposure to fossil fuel-derived PM 10 and susceptibility to COVID-19 that has been reported in epidemiologic studies is biologically plausible.

FIG 2 .FIG 3 .
FIG 2. Effect of 5% CSE on ACE2 expression in A549 cells (A) and purchased (from Promocell) HPNEpCs (B).Results are expressed as mean fluorescence intensity (MFI) adjusted for isotypic antibody control.Columns represent medians from at least 4 separate experiments, and data are compared by the Mann-Whitney U test.
Effect of the antioxidant N-acetyl cysteine on expression of curbside PM 10 -stimulated ACE2 expression (MFI) in purchased HPNEpCs.Columns represent medians from at least 4 separate experiments.Data were compared by either the Mann-Whitney U test or Kruskal-Wallis test and Dunn multiple comparisons test.
b-galactosidase-conjugated antibody (catalog no.2040-06, FIG1.Effect of PM 10 on ACE2 expression.PM 10 was collected from the curbside of Marylebone Road (curbside PM 10 ) and the exhaust of a diesel car (diesel PM 10 ) by using the same cyclone.Cells were cultured with PM 10 for 2 hours, and ACE2 expression was determined by flow cytometry.The results are expressed as mean fluorescence intensity (MFI) adjusted for isotypic antibody control.A, Dose-response relationship between curbside PM 10 and ACE2 expression by A549 cells.Two data points are omitted for visual convenience.B, Replication of A549 dose-response data using 10 mg/mL of curbside PM 10 .C, Effect of curbside PM 10 and diesel PM 10 (10 mg/mL) on ACE2 expression (MFI) in purchased (Promocell) HPNEpCs.D, Effect of curbside PM 10 (10 mg/mL) on ACE2 expression in primary nasal epithelial cells obtained from a local adult donor HPNEpCs.E, Effect of curbside PM 10 (10 mg/mL) on ACE2 expression (MFI) in Calu3 cells.F, Effect of curbside PM 10 on SARS-CoV-2 infection of Vero-E6 cells.A, SARS-CoV-2 CPE expressed as infection enhancement (percentage) compared with the control medium.Data from 7 separate experiments are compared by the Kruskal-Wallis test and Dunn multiple comparisons test.Effect of curbside PM 10 on SARS-CoV-2 viral gene expression in Vero-E6 cells: ORF 1ab (B), S protein (C), and N protein (D).Viral protein data from 5 separate experiments are expressed as FFU/mL calculated from a standard curve with an input of 1.5 3 10 6 FFU/mL and serial 10-fold dilutions of SARS-CoV-2 viral stock.Ct values are normalized to the standard curve for quantification.Columns represent medians from 5 separate experiments, and data are compared by using the Mann-Whitney U test.